histology.umsa.edu.ua · 2020. 12. 31. · 1 subject histology, cytology and embryology modul №1...

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Subject Histology, cytology and embryology

Modul №1 Cytology, embryology and basic histology

Submodul №1 Cytology

Topic 1 THE SUBJECT OF HISTOLOGY METHODS AND

MICROSCOPY Course 1

Faculty Dental

Hours: 2

1. The topic basis: the topic “The subject of histology” is very important for future

doctors in their professional activity, positively influences the students in their

attitude to the future profession, forms professional skills and experience as well as

taking as a principle the knowledge of the subject learnt.

2. The aims of the training course:

1) To have general knowledge of the topic studied.

2) To understand, to remember and to use the knowledge received.

3) To learn the classification, structure and functions of the different

histological methods.

4) To form the professional experience by reviewing, training and

authorizing it.

5) To be able to carry out laboratory and experimental work.

3. Materials for the before – class work self – preparation work:

3.1 Basic knowledge, experience, skills necessary for studying the topic in

connection with other subjects:

To know To be able to

Med. Biology the structure of the cells and

tissues

work with a light

microscope

Med. Physics the structure of the light and

electron microscopes

work with a light

microscope

Organic Chemistry the chemical content of the cell Speak of the topic

The contents of the topic:

Medical histology applies microscopy to the human body, seeking to discover the

nature of its smaller structures, how they relate to each other, and what they do.

Thinking in histology runs along these lines.

Histology is colourful. Almost everything seen is actually there; which is not to say

that what is not seen is absent. One handles and views actual slides - the source

material for most of histology, not just someone else's selected images. The

structures can be interpreted as parts in developmental and functional sequences,

and be fitted together by satisfying accounts, for example, of how cells defend the

body. So much is now known of the roles of cells and structures that histology is

both descriptive microanatomy, and an introduction to function for the whole body.

Preparation of the Material

1. A major distinction can be drawn between dead and living preparations Dead

Section - a thin slice of tissue or organ - on a glass slide or metal grid.

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Smear on a glass slide - suitable for suspensions, e.g., blood, urine, mucus, cyst

fluid, bone marrow, etc.

Spread sheet of tissue stretched thin, e.g., areolar connective tissue.

Teased apart fibrous elements, e.g., muscle.

Living

Such preparations may be out of the body in a tissue culture system, or

living within the body but in an observable situation, e.g., a transparent

chamber inserted into the ear or skin.

The first need is to keep the preparation alive. This seriously limits the

facilities for observation. For example, staining is usually impracticable.

Thus, phase-contrast or dark-field microscopy has to be used in order to

overcome the poor contrast between natural structures.

2. Steps needed to make and study a histological section

1. Fixation to prevent post-mortem decomposition, preserve structure, and intensify subsequent staining.

2. (a) Steps involved in imbedding the tissue in a block of wax or plastic, or (b) freezing of the material to a firm mass, for cutting into thin sections on a

microtome; 1-150 microns (µm) thick for light microscopy (LM); 30-60

nanometres (nm) for electron microscopy (EM).

3. Units: based on the metre (m): micron/micrometre (µm) = 10-6m; nanometre (nm)/ millimicron (mµ) = 10-9m; Ångström (Å) = 10-10m; 10Å=1nm.

4. Mounting of the section on a glass slide or metal grid. Staining of the section with one or more reagents, e.g., solutions of metallic salts, in one or more

stages.

5. For light microscopy, the removal of surplus stain and water, and steps involved in holding a thin glass coverslip to the section with a mounting

medium having a refractive index close to that of glass.

6. Observation and recording by means of the microscope, and notes, photomicrography, projection drawing, labelled sketches, counting and

reconstructions, digital and videorecording. A drawback to using our eyes as

part of the observing instrument is that the visual system does not record

accurately.

Microscopy

1. Microscopy in general

The main distinction is between light microscopy and electron microscopy. The

usual light microscope uses a visible light source with a system of condenser lenses

to send the light through the object to be examined. The image of this object is then

magnified by two sets of lenses, the objective and the eyepiece. Total

magnification is then the product of these two lens systems, e.g., 40 X 10 = 400.

The resolution or resolving power - how close two structures can be and still be

seen as separate - is a measure of the detail that can be seen, and for the light

microscope is about 0.25 µm. This limit to resolution is determined mostly by the

wavelength of the light; and, however powerful the lens, 0.25 µm cannot be

improved upon.

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The only way to improve resolving power is to reduce substantially the wavelength

of the light. This is achieved by the electromagnetic beam of the electron

microscope. The beam is focused through the object suspended on its metal grid,

and is magnified before striking a fluorescent screen to be transformed into a

visible image.

The resolutions so far achieved in biology with transmission electron microscopy

(TEM/EM) are of the order of 1 nm at a magnification of X 2 000 000. The other

forms of microscopy - phase-contrast, interference, fluorescence, confocal

scanning, atomic-force (and X-ray diffraction) - will be discussed in , in relation to

the problems for which they are suited.

2. Microscopy for the student

1. The usual class microscope has eyepieces/oculars magnifying X 7, and an objective nosepiece carrying X 8, X 20, X 40, and X 90 (oil immersion)

lenses. Normally the three lower-power lenses are kept mounted on the

nosepiece, whilst the oil immersion objective may be mounted or kept

separately.

2. Every time it is used, the microscope should be set up to the best optical advantage. How to do this is described briefly below.

3. Keep in mind the limit to resolution. In practical terms, make special note of those structures that need an oil immersion lens to be seen or are visible only

in electron microscopy.

4. The section has some thickness, so that the fine-focusing adjustment should be used continually during observation to bring out fine detail, e.g., cilia on

cells. Essentially, though, we are getting a two-dimensional picture from an

originally three-dimensional piece of material. For what the structure looked

like in the third dimension, the student can try to reconstruct mentally what

is going on in the missing dimension, and look up views of the structure in

scanning electron microscopy.

5. Artefacts (appearances not due to the original nature of the material as

obtained from the body) can arise at all stages in the treatment of the section.

Gross examples arise from: (1) clumsy excision from the body; (2) poor or

inappropriate fixation; (3) shrinkage and, worse, uneven shrinkage, leading to

artificial spaces and distorted relations; (4) cutting scores from a bad microtome

knife; (5) the section not flat on the slide; (6) water, dirt or bubbles on or in the

section; (7) dirt on the microscope lenses; (8) patchy or faded staining;

unbalanced staining when more than one stain has been applied; (9) precipitate

from fixative or stain; (10) tears and folds in the section.

6. Setting up the microscope – see in the album. 3. Differences between light and electron microscopy

1. Table 1 gives some differences between the two approaches. The detailed morphology revealed by EM may be called fine or submicroscopic

structure/ultrastructure.

2. The direct comparison of LM and EM images of a structure requires that the magnifications be of the same order. Noting the magnification, on the 'scope

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or in the figure legend, allows one to adjust one's expectations of what may

be seen, and should always be done.

3. A growing practice in histology and pathology is to fix and prepare the tissue by EM standards, imbed in plastic and cut semi-thin (1 µm) sections

for staining by modified LM methods. LM then reveals good cellular detail

and fewer artefacts.

4. Two other techniques yield anatomical images - fibre-optic endoscopy and scanning EM - are being digested by the anatomical texts. Endoscopy from

its low magnification is marginal to histology, but related in that endoscopy

is used to obtain biopsy specimens for histopathology.

SEM strengthens one's conception of microscopic structures, e.g., cilia, renal

podocytes, bone under resorption, and effectively counters the unavoidable

impression of structures existing only in two dimensions.

Table 1. Some differences between light and electron microscopy.

Light microscopy Electron microscopy

Image is presented directly to the eye.

Image keeps the colours given the

specimen by staining.

Image is in shades of green on the screen;

photographically, only in black and

white.

Modest magnification to X 1500; but a

wider field of view and easier

orientation.

High magnification, up to X 2,000,000

thus the range of magnification is greater.

Resolving power to 0.25 µm. Resolving power to 1 nm (0.001µm.)

Frozen sections can yield an image

within 20 minutes. Processing of tissue takes a day at least.

Crude techniques of preparation

introduce many artefacts.

(Histochemical methods are better.)

High resolution and magnification

demand good fixation (e.g. by vascular

perfusion), cleanliness and careful

cutting, adding up to fewer artefacts.

Section thickness (1-30 µm) gives a

little depth for focus for appreciation of

the third dimension. Serial sections can

be cut, viewed and used to build a

composite image or representation.

Very thin sections provide no depth of

focus, but 3-D information can be had

from: (a) thicker sections by high voltage

EM; (b) shadowed replicas of fractured

surfaces; (c) scanning electron

microscopy (SEM).

Most materials and structures cannot be

stained and viewed at the same time;

stains are used selectively to give a

partial picture, e.g. a stain for mucus

counterstained to show cell nuclei.

Heavy metal staining gives a more

comprehensive picture of membranes,

granules, filaments, crystals, etc.; but this

view is incomplete and even visible

bodies can be improved by varying the

technique.

Specimen can be large and even alive.

Specimen is in vacuo. Its small size

creates more problems with sampling and

orientation.

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Dynamic nature of the cell.

The cell is not a static entity in life. Its chemical constitution and morphology are

in continuous flux. Its complement of organelles is altering, with wearing-out and

replacement, i.e., the cell is having to synthesize its own material. The cell itself

represents a system of activities isolated to partial extent from an extracellular

environment. Within the cell things are constantly being altered, moved around and

joined up within the membranes. The membranes define temporary compartments

separated from the cytoplasm, where particular activities can be confined and

controlled by enzymes attached to the extensive membrane surfaces. Dynamic

aspects of the cell's existence are partly deduced from a study of its morphology in

specimens fixed in various states, partly from microscopical observations of living

cells, and from histophysiological experiments outlined in.

➢ Biological tissues must undergo a series of treatments to be observed with light and electron microscopes. The process begins by stabilization of the tissue with

chemical fixatives. Next, the tissue is made rigid to allow sectioning. Finally, it is

stained to provide contrast for visualization in the microscope.

➢ Steps in tissue preparation 1. Fixation 2. Dehydration 3. Infiltration and embedding 4. Sectioning 5. Staining 6. Chemical Fixation

➢ Preserves cellular structure and maintains the distribution of organelles.

➢ Formaldehyde and glutaraldehyde are the most commonly used chemical fixatives. They stabilize protein by forming cross-links between primary amino

groups. Formaldehyde in solution is referred to as formalin.

➢ Osmium tetraoxide is a fixative used to preserve lipids, which aldehydes cannot do. Osmium combines with and stabilizes lipid and, in addition, also adds a brown

color (light microscopy) or electron density (electron microscopy) at the site of the

lipid. Osmium fixation is required for electron microscopy, especially to preserve

the lipid in membranes.

DEHYDRATION, INFILTRATION, AND EMBEDDING

➢ Tissue water is not miscible with the embedding solutions and must be replaced using a series of alcohols at increasingly higher concentrations. This step is

followed by alcohol replacement with an intermediate solvent that is miscible with

both alcohol and the embedding solutions.

➢ Infiltration and embedding. The liquid form of the embedding compound, for example, paraffin wax or epoxy plastic, replaces the intermediate solvent. The

liquid embedding medium is allowed to solidify, thereby providing rigidity to the

tissue for sectioning.

Sectioning

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➢ The embedded tissue is cut thin enough to allow a beam of light or electrons to pass through.

➢ Section thickness

Light microscopy. 1–20 microns

Electron microscopy. 60–100 nanometers

➢ Section planes 1. Cross-section (cs) or tranverse section (ts) is a section that passed

perpendicular to the long axis of a structure.

2. Longitudinal section (ls) is a section that passed parallel to the long axis of a structure.

3. Oblique (tangential) section is any section other than a cross- or longitudinal section.

STAINING

➢ Most tissues have no inherent contrast; thus, stains must be applied to visualize structures.

➢ Conventional staining. Relies mostly on charge interactions. 1.Light microscopy

Hematoxylin and eosin (H&E). These two dyes are the most commonly used stains in routine histology and pathology slides. Most conventional stains

bind to tissue elements based on charge interactions, that is, positive charge

attraction for a negatively charged structure. Hematoxylin binds to

negatively charged components of tissue, the most prominent being nucleic

acids. Hematoxylin imparts a purple/blue color to structures and, therefore,

the nucleus and accumulations of rough endoplasmic reticulum in the

cytoplasm, which contains large amounts of nucleic acid, appear blue or

purple in sections.

Structures, like the nucleus and rough endoplasmic reticulum that stain with hematoxylin, are referred to as basophilic or “base loving.” The term

basophilia, refers to the property of a structure or region that stains with a

basic dye, such as hematoxylin. Structures that stain with eosin, for example,

the cytoplasm of most cells and collagen fibers, appear pink or orange and

are referred to as eosinophilic.

2.Electron microscopy

Images in the electron microscope are produced by passing a beam of

electrons though the tissue that has been “stained” with salts of heavy

metals, usually lead (lead citrate) and uranium (uranyl acetate). These metals

bind to areas of negative charge and block the passage of the electrons

through the section, resulting in a dark area in the electron micrograph.

Electron density is also achieved using osmium tetroxide, which also serves

as a lipid fixative.

Areas or structures in tissue that bind the metals are referred to as electron dense. Areas where the metals do not bind appear light and are referred to

electron lucent.

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➢ Histochemical staining. Localizes chemical groups

Osmium tetroxide. Stains lipids

Periodic acid–Schiff stain (PAS). Stains carbohydrates

Staining Reactions

Staining reactions have both physical and chemical characteristics. The

mechanisms involved in staining include the following:

The dye may actually be dissolved in the stained substance. Most fat staining is

accomplished in this fashion. A dye may be absorbed on the surface of a structure,

or dyes may be precipitated within the structure, simply because environmental

factors (pH, ionic strength, temperature, etc.) favor absorption or precipitation.

Most staining reactions involve a chemical union between dye and stained

substance through salt linkages, hydrogen bonds, or others. Staining with these

dyes results in a predictable color pattern based in part on the acid base

characteristics of the tissue. However, color and color distribution are not

absolutely reliable for discrimination between tissue components. Color will vary

not only with specific stains used, but also with the conditions that exist during

preparation of the slide. These include everything from the initial fixing solution to

the ionic strength of the staining solution and the differentiating solvents utilized

after staining.

Acid and Basic Dyes

Most histologic dyes are classified either as acid or as basic dyes. An acid dye

exists as an anion (negatively charged) in solution, while a basic dye exists as a

cation (positive charge). For instance, in the hematoxylin-eosin stain (H&E), the

hematoxylin-metal complex acts as a basic dye. The eosin acts as an acid dye.

Any substance that is stained by the basic dye is considered to be basophilic; it

carries acid groups which bind the basic dye through salt linkages. When using

hematoxylin, basophilic structures in the tissue appear blue (or purple or brown;

this varies according to the stain that is being used). A substance that is stained by

an acid dye is referred to as acidophilic; it carries basic groups which bind the acid

dye. With eosin, acidophilic structures appear in various shades of pink. Since

eosin is a widely used acid dye, acidophilic substances are frequently referred to as

eosinophilic.

Trichrome Stains

In the trichrome stains, which commonly employ more than one acid dye, use is

made of dye competition. For instance, acid fuchsin and picric acid are used in Van

Gieson's trichrome stain. In the picric acid-fuchsin mixture, the small picric acid

molecule reaches and stains the available sites in muscle before the larger fuchsin

molecules can enter. Used by itself, acid fuchsin has no difficulty in staining

muscle.

Neutral Stains

These are compounds of an acid dye and basic dye. For instance, aqueous

solutions of acid fuchsin may be neutralized by addition of aqueous methyl green.

The resulting neutral product is water insoluble, but may be kept in solution by the

presence of excess amounts of either component. The tissue stained with such a

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solution may show affinity for the acid dye, the basic dye, and for the whole

compound. Some blood stains are "neutral stains." Wright's Stain, for instance, is

formed by the combination of partially oxidized and demethylated methylene blue

and eosin. Such a stain can be used to differentiate between blood cells that contain

acidic, basic, and neutral granules.

Hematoxyl and Eosin (H&E)

This is a good general stain and is widely used. Most of your slides are stained

with H&E. A hematoxylin-metal complex acts a as a basic dye, staining nucleic

acids in the nucleus and the cytoplasm blue, brown, or black. Eosin is an acid

aniline dye which stains the more basic proteins within cells (cytoplasm) and in

extracellular spaces (collagen) pink to red. Cartilage and mucus may stain light

blue.

Masson Trichrome Stain

A staining sequence involving iron hematoxylin, acid fuchsin, and light green. It is

a good stain for distinguishing cellular from extracellular components. Collagen

fibers stain an intense green. Black or brown nuclei; mucus and ground substances

take on varying shades of green. Cytoplasm stains red. Elastic fibrils, red blood

cells and nucleoli stain pink.

Aldehyde Fuchsin

Stains elastic fibers purple to black. Can be counter-stained with a dye of

contrasting color, such as metanil yellow.

Verhoeff's Hematoxylin

Another variant of the versatile hematoxylin stains. This method stains elastic

fibers black in addition to nuclei.

Reticular Fiber Stain – Weigert

Reticular fibers are impregnated with a silver salt and appear as sharp black.

Collagenous fibers usually stain purple. This stain can be used with a counterstain

or without, if the silver stain turned out very dark.

Wright's/Giemsa Stain

This and similar stains for blood and bone marrow smears are mixtures of basic

(methylene blue derivatives) and acid dyes (usually eosin). According to the

number of acid and basic groups present, cell components take up the dyes from

the mixture in various proportions. Some blood stains use acid and basic dyes in

separate dye baths.

Metachromatic Stain

Certain basic dyes, such as toluidine blue, stain nucleic acids blue (the

orthochromatic color), but sulfated polysaccharides purple (the metachromatic

color). When dye molecules bound to sulfate groups are stacked closely together,

the dye experiences a color shift from blue to purple. Thus, a metachromatic

reaction often indicates the presence of numerous closely packed sulfate groups.

Plastic Sections Stained with Toluidine Blue or with H&E

Plastic embedded tissues can be cut as thin as 0.1 um with a glass knife. These

sections are then stained with toluidine blue in an alkaline solution. Almost all

tissue components are stained more or less deeply (usually a bluish-purple) and

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structural detail is very sharp. For the knowledgeable observer, this type of

preparation may be very informative.

Periodic Acid Schiff (PAS)

Adjacent hydroxyl groups (1, 2 glycols) or amino and hydroxyl groups are

oxidized to aldehyde groups with periodic acid. Schiff's Reagent then produces a

red or magenta addition product with the aldehyde groups and this technique

identifies a number of polysaccharides and carbohydrate-containing compounds.

The slide may also be counter stained with hematoxylin. Feulgen Reaction: Mild

hydrolysis with HCl frees the aldehyde group of deoxyribose, which is then reacted

with the Schiff's reagent. This reaction is highly specific for DNA and may also be

used with a counter stain for the cytoplasm.

➢ Immunocytochemistry. Localization of specific antigens in cells using labeled antibodies

➢ In situ hybridization. Detection of messenger RNA or genomic DNA sequences using labeled nucleotide probes

ARTIFACT

➢ The term artifact is used to refer to any feature of a tissue section that is present as a result of the tissue processing. These include tears and folds, shrinkage, spaces

resulting from extracted cellular contents (e.g., lipid, precipitates), and

redistributed organelles.

MICROSCOPY

➢ Properties 1.Resolution is the smallest degree of separation at which two objects can still be

distinguished as separate objects and is based on the wavelength of the

illumination.

Light microscopy. Approximately 200nm

Electron microscopy. Approximately 1nm 2.Magnification. Enlargement of the image

➢ Bright field microscope

An image is formed by passing a beam of light through the specimen and then focusing the beam using glass lenses.

The bright field microscope is called a compound microscope because it uses two lenses, objective and ocular, to form and magnify the image. The

compound microscope typically has a total magnification range of 40–1000

times.

➢ Electron microscope 1.Transmission electron microscope (TEM)

An image is formed by passing a beam of electrons through the specimen and focusing the beam using electromagnetic lenses.

Similar arrangement of lenses is used as with optical microscopy; magnification is up to 400,000 times, which is sufficient to visualize

macromolecules (e.g., antibodies and DNA).

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2.Scanning electron microscope (SEM). The image is formed by electrons that are

reflected off the surface of a specimen, providing a three-dimensional image;

magnification ranges from 1–1000 times.

➢ Freeze fracture technique

This technique is used to examine the number, size, and distribution of membrane proteins.

A tissue is frozen and mechanically fractured; the exposed membrane surface is coated with a thin metal film called a “replica.”

The replica is viewed by TEM. Membrane proteins appear either as bumps or pits in the replica.

SECTION INTERPRETATION

➢ In histology, three-dimensional tissues are viewed in two dimensions; therefore, it is extremely important to learn to visualize the threedimensional structure from

the two-dimensional image. For example, a cross-section through a tubular

structure appears as a ring, whereas a longitudinal section appears as two parallel

bands. As an added challenge, most sections pass obliquely to these perpendicular

axes and, thus, require further “mental gymnastics.”

UNITS OF MEASURE

➢ Millimeter (mm) = 1/1000 meter, 10-3M

➢ Micron, micrometer (mm) = 1/1000mm, 10-3mm, 10-6M

➢ Nanometer (nm) = 1/1000mm, 10-3mm, 10-9M

➢ Еngstrцm unit (Е) = 1/10nm, 10-10M Cytological description of an individual cell.

In light microscopy involves: (1) relative and absolute size; (2) shape; (3) number

of nuclei; (4) shape and size of nucleus/nuclei; (5) intensity of nuclear staining; (6)

amount of cytoplasm; (7) staining affinity of cytoplasm, e.g., basophilic,

acidophilic (eosin), argentophilic (silver stains), or chromophobe (liking no stain);

(8) granular cytoplasm; (9) nature of any inclusions, for instance, melanin pigment,

fat, carbon, bacteria, zymogen granules, glycogen, mucus; (10) specializations of

the cell membrane, e.g., cilia, a brush/striated border (many microvilli); (11)

distinctive organelles in cytoplasm and their position, e.g., prominent Golgi

complex, many fibrils, numerous orderly mitochondria giving another striated

effect, Nissl substance (GER) in nerve cells; (12) whether the cell is in some phase

of mitosis or meiosis; (13) the cell's surroundings; (14) manifest properties of the

living cell, e.g., motility, phagocytosis, contractility.

3.3. Literature recommended

Main Sources:

1. L.C. Junqueira, J. Carneiro - “Basic histology” – 11 edition - 2005. 2. A.S. Pacurar, J.W. Bigbee – “Digital histology” – Verginia - 2004. 3. “Color Atlas of basic histology” – R.Berns – 2006. 4. Sadler T.V. – “Medical embryology” Montana – 1999. 5. Ronald W., Dudek Ph.D. –“Embryology” 2 edition – 1998. 6. Inderbir Singh Textbook of Human Histology.- Jaypee. India. - 1997.

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7. Ten Cate A.R. Oral Histology.- St.Louis, Baltimore, Toronto.- 1995. 8. William A., Beresford M.A. Cytology and Histology.- Anatomy department,

West Virginia University, USA.- 1992.

9. K.E. Jonson - “Histology and cell biology” – 2 edition – Washington –

1991.

Additional ones:

1. Methodical Instructions.

3.4 How to work with the literature recommended:

Main tasks Recommendations

To review the material

To learn the material

To read and compose the plan

To answer the questions

To do the test on the material

To be ready to answer the topic

To use the material studied

To use the material on pages

To learn the new material and be

ready to write a summary

To be ready to give an answer to

the following:

3.5. Self-control material:

A. Questions to be answered:

1. Technique of light microscopy. 2. Special methods of light microscopy. 3. Trasmission and sweepable electronic microscopy. 4. Methods of research of living cages and fabrics Intravital painting. 5. Research of living cells and fabrics is in a culture (in vitro). 6. Research of separate cells and their cultivation. 7. Іmmunofluorescent, methods of radioautographic research. 8. Modern methods of study of cellular content. 9. Quantitative methods of research. 10. Basic principles of making of preparations for a light microscopy. 11. Fixing. Types of fixings. Method of making of cuts. 12. Dehydration, compression and inundation. 13. Colouring and contrasting. 14. Classification of histological dyes to on by chemical properties 15. Conclusion of histological preparations 16. Types of microslides are a cut, stroke, imprint, tapes, microsection. 17. Methods of analysis of image of cellular and tissue structures 18. Prizhiznennye methods of research. 19. Intravital and supravital painting. B. Test tasks to be done: Tests are applied

4. Self-preparation in the classroom.

1) Listen to the information.

2) Work with the tables and a Light microscope.

3) Ask about the problems that haven’t been found in the information given.

4) To sketch in the album the investigated preparations.

5. Self-preparation work at home.

1) Review the material learnt in the classroom.

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2) Compose the plan of your answer.

3) Answer the questions to this topic.

4) Do the test given above.

6. The subject of the research work.

- “The History of Histology”. - “New methods in histological research”.




Topic 2 CELLS. PLASMALEMMA. CELL JUNCTIONS

Course 1

Faculty Dental

Hours: 2

1. The topic basis: the topic “Plasmalemma. Cell junctions.” is very important for

future doctors in their professional activity, positively influences the students in

their attitude to the future profession, forms professional skills and experience as

well as taking as a principle the knowledge of the subject learnt.




3) To learn the classification, structure, functions of cell’s surface.


authorizing it.






Anatomy - -

Histology - -

Med. Biology the structure and functions

of cell’s surface

work with the light

microscope



work with the light

microscope

Organic Chemistry Chemical content of the cell Speak on the subject

3.2.The contents of the topic:

1. Body components

The human body content of the cells, tissues, organs and organ’s systems. These

are cells, extracellular substances, and body fluids. Fluids can have their suspended

solid constituents viewed microscopically as smear preparations (see Blood), but

are otherwise of limited histological interest. Extracellular substances are

important for the cells whose environment they form: they reflect and help control

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cellular activities, aside from their critical structural mechanical properties.

Individual materials can be seen and localised by histochemistry.

2. Cells: chemical constitution and fixation

1. Composition: much water; proteins, nucleic acids, lipids, carbohydrates,

amino acids, minerals, hormones, vitamins, etc.

2. Fixation stabilizes mainly proteins, and protein conjugates. These substances are used as building materials for the firmer structures of the cell. Lipids,

minerals, glucose, and smaller molecules are usually lost from the section

during processing. What is left is a skeleton of structures - membranes,

granules, filaments - made up of proteins, polypeptides, polysaccharides and

some other macromolecular materials. The special steps of histo- and

cytochemistry serve and reveal some smaller molecules.

3. Cells: living properties and specialization

1. Properties of cells: (a) general - communication, respiration and energy storage and release, synthesis, excretion, growth, differentiation,

reproduction; (b) specialized - irritability to stimuli (excitability), motility,

contractility, conductivity, absorption, phagocytosis, secretion.

2. During development from the fertilized oocyte, a great variety of cells is formed in the mammal, each kind specializing in a certain function, e.g.,

secretion, but many activities, such as energy production, are common to all

cells. The cells of the four primary tissues - epithelial, connective, nervous

and muscular - are divided along lines of specialized function, e.g., muscle

for contractility and excitability.

4. Cell morphology

1. Cells performing a given function have a characteristic size, form and fine structure adapted to that task. However it may help at this stage to think in

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terms of a composite cell having all the features the various cells of the body

display.

2. The cell is defined as a distinct entity by having a thin skin or plasmalemma/cell membrane separating off from the outside a soft, viscous,

almost fluid cytoplasm, in which are suspended a number of firmer,

recognizable structures - organelles and inclusions - and one or more nuclei.

The nucleus, likewise, is a mass of material enclosed in nuclear membranes.

5. Cell components – cytoplasm, plasmalemma and nucleus.

1.Cytoplasm. It consist of organelles, includings and hyaloplasm - the so-called

soluble phase of the cell, consisting mostly of water, dissolved solutes, and

larger molecules in suspension tending to link repetitively with covalent bonds

giving the cytoplasm a dense, viscous colloidal sol or gel consistency.

2.Cell or plasma membrane/plasmalemma

o Made of unit membrane roughly 7.5 nm wide; in the EM picture with

appropriate fixation it has a trilaminar appearance of two dark bands with a

light layer betwen them; the light layer is mostly non-polar lipid, the dark

layers mostly the charged ends of the lipid molecules with attached protein.

o A coat of glycoproteins (sugars+protein) - glycocalyx - adheres to the

outside of most cells – undermembranous complex.

o The plasma membrane is flexible, semi-permeable, and experiences active

transport and potential differences across it.

o By the cell membrane's fusing with the intracellular membrane around

stored material, then breaking open at a point along the line of fusion the

material can be released to outside the cell - exocytosis/emiocytosis. By

bringing extracellular things into an invagination, followed by selective

membrane fusion and separation, materials are brought into the cell -

endocytosis: phagocytosis for solids, pinocytosis for fluids; endocytosis

includes these bulk movements, but also signifies the internalization of

membrane receptors and bound ligands on a smaller scale.

o Specializations of form shown by the plasma membrane include: microvilli,

cilia with basal bodies, (iii) pinocytotic vesicles/pinosomes/caveolae

intracellulares, infoldings or plications, desmosomes, gap junctions/nexuses,

occluding junctions, stereocilia (long microvilli).

o Some vesicles involved in transport into the cell have prominent coats of

clathrin attached to the membrane.

o Proteins of internal surface of the plasmalemma form the

cytoskeleton(submembranous complex)

3.Cell nucleus consist of Nuclear membrane, Chromatin, Nucleoplasma and

Nucleola.

15

6. Cell Membranes

I. Functions of the membrane are:

1. Separation the cell from the external enviropment. 2. Firm attachment to other cells or a basal lamina; membrane specializations

for this are: junctional complexes, gap junctions/ nexuses, desmosomes,

hemidesmosomes, intercalated discs, and membrane interdigitations.

3. Transport of materials in and out of the cell served by: permeability (selective) of the membrane, active transport through the membrane,

endocytosis, and its more scaled up forms - pinocytosis and phagocytosis,

exocytosis; and increased exchange surface area by microvilli (thousands on

a cell), and infoldings of membrane.

4. Movement of the cell itself by pseudopodial, filipodial, or lamellipodial extensions (think karate: fist, finger, or side of the hand) and the release of

any firm attachments, or by flagellate activity, e.g., by sperm. (Microspikes

and ruffles are alternative names for filopodia and lamellipodia,

respectively.)

5. Movement of materials outside the cell by the activity of cilia, e.g., ciliated epithelia of the respiratory tract and uterine tube. The wide-spread

occurrence of solitary cilia (flagella), e.g., on neurons, adrenal cells, smooth

muscle, may involve a vestigial body or one still functional. The stereocilia

of the male reproductive tract are non-motile, clumped, long microvilli,

probably absorptive.

6. Communication and transduction. Each cell collaborates with both adjacent cells, and those of the whole body, for development, growth,

homeostasis, regeneration, and its own particular task. The importance of the

cell membrane in receiving and sending the necessary signals is stressed by

the number of examples given:

The binding of hormones to receptors on the membrane.

The binding of the lymphocyte's membrane receptor to an antigen.

Transmitter chemicals depolarize neurons and muscle cells.

Excitable tissues propagate action potentials along the membranes.

16

Schwann cells wrap their membranes many times round an axon's to make

myelin sheath segments for faster signalling.

Chemical stimuli are transduced into nerve impulses in chemoreceptors;

mechanical stimuli in mechanoreceptors.

Gap junctions permit ions and excitation to spread from cell to cell, and

unify and synchronise actions of many cells/cell assemblies.

In development, epithelial and mesenchymal cells interact in sequence to

induce cell differentiations, e.g., in tooth and glands.

Cells attract and fuse with one another to form multinucleated cells, e.g.,

skeletal muscle and osteoclasts.

Chemotactic agents act on phagocytic cells to attract them to their targets.

Keratinocytes of the skin phagocytose melanin pigment offered to them in

the processes of melanocytes.

Macrophages detect spent or abnormal red blood cells, and hold and engulf

either the whole cell, or the part holding an unwanted body.

II. Molecules Wherever such actions are described, special molecules are acting,

by binding to each other, changing their conformation, or some other means.

o Spectrin/fodrin provides a subplasmalemmal skeleton attached to the cell

membrane by ankyrin, and to actin of the cytoskeleton, to permit control of the

membrane's shape and movement.

o Cell adhesion molecules (CAMs) allow cells to attach to only certain cell

types.

o Integrins are cell-surface-membrane dimeric molecules (an alpha with a

beta), by which cells choose to which extracellular matrix (ECM) components

they wish to fasten, e.g., laminin.

o Connexins are proteins that combine as hexamers to form connexons - the

gap-junction channels, allowing ions and small molecules to pass between

cells. Connexins and the transports allowed vary among liver cells, neurons.

o Occludins are responsible for the seal preventing passage of materials past

inter-epithelial tight junctions.

7. Basic Membrane structure

17

The proteins of the membrane are: integrals, semi-integrals (external and internal)

and peripheral (external and internal). o They may form are esssential part of the structure of the membrane

i.e., they may be structural proteins. o Some proteins play a vital role in transport across the membrane and

act as pumps. Ions get attached to the protein on one surface and

move with the protein to the other surface. o Some proteins are so shaped that they form passive channels through

which substances can diffuse through the membrane. However, these

channels can be closed by a change in the shape of the protein. o Other proteins act as receptors for specific hormones or

neurotransmitters. o Some proteins act as enzymes.

8. Contacts between adjoining cells In tissues in which cells are closely packed the cell membranes of adjoining cells

are separated, over most of their extent by a narrow space (about 20 nm). This

contact is sufficient to bind cells loosely together, and also allows some degree of

movement of individual cells. In some regions the cell membranes of adjoining

cells come into more intimate contact: these areas are marked by structural

specializations as described below.

Zonula Occludens At such a junction the two plasma membranes are in actual contact. These

junctions act as barriers that prevent the movement of molecules into the

intercellular spaces. For example, intestinal contents are prevented by them from

permeating into the intercellular spaces between the lining cells. Zonulae

occludens are, therefore, also called tight junctions. Apart from epithelial cells,

zonulae occludens are also present between endothelial cells. In some situations

18

occlusion of the gaps between the adjoining cells maybe incomplete and the junc-

tion may allow slow diffusion of molecules across it. These are referred to as leaky

tight junctions.

Desmosomes (Maculae Adherens)

This is the most common type of junction between adjoining cells. A desmosome

is a small circumscribed area of attachment. At the site of a desmosome the plasma

membrane (of the cell) is thickened because of the presence of a dense layer of

protein on its inner surface (i.e., the surface towards the cytoplasm). The thickened

areas of the two sides are separated by a gap of 20 nm or more. The region of the

gap is rich in a glycoprotein called desmoglea. The thickened areas of the two

membranes are held together by fibrils that appear to pass from one membrane to

the other across the gap. Closer examination shows, however, that the fibrils do not

pass from one cell to the other. Instead the fibrils of each side are in the form of

loops: the loops of the opposing membranes interlock. The cytoplasmic aspect of

the thickened areas of the cell membrane also gives attachment to numerous fibrils

that pass into the cytoplasm. Desmosomes are present where strong anchorage

between cells is needed. Zonula Adherens In some situations, most typically near

the apices of epithelial cells, we see a kind of junction called the zonula adherens.

This is similar to a desmosome in being marked by thickenings of the two plasma

membranes, to the cytoplasmic aspects of which fibrils are attached. However, the

junction differs from a desmosome in that instead of being a small circumscribed

area of attachment the junction is in the form of a continuous band around the

apical part of the epithelial cell; and in that the gap between the thickenings of the

plasma membranes of the two cells is not traversed by filaments. An adhesive

material is probably present in this situation. Apart from epithelial cells zonulae

adherens are also seen between smooth muscle cells, and between myocytes of

cardiac muscle in the region of intercalated discs. Gap Junctions (Nexuses) At

these junctions the plasma membranes are not in actual contact (as in a tight

19

junction), but lie very close to each other, the gap being reduced (from the normal

20 nm) to 3 nm. Placed in this gap there are bead-like structures arranged in the

form of hexagons. A minute canaliculus passing through each 'bead' connects the

cytoplasm of the two cells thus allowing the free passage of substances from one

cell to the other. Gap junctions are, therefore, also called maculae comnumicantes.

They are widely distributed in the body. Junctional Complex Near the apices of

epithelial cells the three types of junctions described above, namely zonula oc-

cludens, zonula adherens and macula adherens are often seen arranged in that

order. They collectively form a junctional complex.

Stem cells.

For a stable population, the corollary to cell death is cell renewal. This requires:

o the proliferation of cells;

o an enduring population of stem cells;

o controls (+ & -) that promote division of stem cells to maintain their

numbers - self-replication;

o controls that cause differentiation of certain of the stem cells to

become the determined/committed precursors of the mature cells of

the tissue;

o factors to promote division of the precursors/progenitors and their

further differentiation. The controlling factors include cytokines.

More is known about the ensuing progenitor cells than about the stem cells.

Although not essential to the concept of stem cells, at step (iv) above, stem

cells usually give rise to more than one lineage of differentiated cells, in

order to furnish the needed diversity of cell types in blood and most

epithelia. 3.3. Literature recommended

Main Sources:

1. L.C. Junqueira, J. Carneiro - “Basic histology” – 11 edition - 2005. 2. A.S. Pacurar, J.W. Bigbee – “Digital histology” – Verginia - 2004.

3. “Color Atlas of basic histology” – R.Berns – 2006. 4. Sadler T.V. – “Medical embryology” Montana – 1999. 5. Ronald W., Dudek Ph.D. –“Embryology” 2 edition – 1998. 6. Inderbir Singh Textbook of Human Histology.- Jaypee. India. - 1997. 7. William A., Beresford M.A. Cytology and Histology.- Anatomy department,

West Virginia University, USA.- 1992.

8. K.E. Jonson - “Histology and cell biology” – 2 edition – Washington – 1991.

Additional ones:


3.4. How to work with the literature recommended:








20





To be ready to give an answer to

the following:


A .Questions to be answered:

1.What is the cell?

2. What basic functions of cells do you know?

3. Name the basic components of a cell.

4. What are the basic functions of plasmalemma?

5. What specific membrane proteins distinguish?

6. How is the elementary biological membrane constructed?

7. What are the contacts between adjoining cells?

8. Describe a structure of zonula occludense?

9. Describe a structure of desmosomes (maculae adherens) and zonula adherens.

10. What is nexus?

11. What is the junctional complex?

B. Test tasks to be done: See enclosure.



2) Work with the tables and microscope.


4) To sketch in the album the investigated preparations.







- “The active and passive transport through a cellular wall”. - “The role of intercellular contacts in the preservation of homeostasis”.




Topic 3 ORGANELLES AND INCLUSION

Course 1

Faculty Dental

Hours: 2

1. The topic basis: the topic “Organelles and Inclusion.” is very important for







21

3) To learn the classification, structure and functions of organelles

and inclusion.


authorizing it.






Med. Biology the structure of the cells and tissues work with a light

microscope

Med. Physics the structure of the light and electron

microscopes

work with a light

microscope

Organic Chemistry the chemical content of the cell Speak of the topic

3.2.The contents of the topic:

The cytoplasm of a typical eukaryotic cell contains various structures that

are referred to as organelles. They include the membrane-bound and the non-

membranous organelles. Other components of cytoplasm are gialoplasm and

inclusions.

Mitochondria Mitochondria can be seen with the light microscope in specially stained

preparations. They are so called because they appear either as granules or as rods

(mitos = granule; chondrium = rod). The number of mitochondria varies from cell

to cell being greatest in cells with high metabolic activity (e.g., in secretory cells).

Mitochondria vary in size, most of them being 0.5 to 2μm in length.

The mitochondrion is bounded by a smooth outer membrane within which

there is an inner membrane. The inner membrane is highly folded on itself forming

incomplete partitions called cristae. The space bounded by the inner membrane is

filled by a granular material called the matrix. This matrix contains numerous

enzymes. It also contains some RNA and DNA: these are believed to carry

information that enables mitochondria to duplicate themselves during cell division.

An interesting fact, discovered recently, is that all mitochondria are derived from

those in the fertilized ovum, and are entirely of maternal origin.

22

Mitochondria are of great functional importance. They contain many

enzymes including some that play an important part in Kreb's cycle. ATP and GTP

are formed in mitochondria from where they pass to other parts of the cell and

provide energy for various cellular functions. These facts can be correlated with

the observation that within cell mitochondria tend to concentrate in regions where

energy requirements are greatest. The enzymes of Kreb's cycle are located in the

matrix, while enzymes associated with the cytochrome system are present on the

inner mitochondrial membrane. Mitochondria are also concerned with fatty acid

metabolism, and various other chemical reactions. Endoplasmic Reticulum

The cytoplasm of most cells content with com of membranes that

constitute the endoplasmic reticulum. The membranes form the boundaries of

channels that may be arranged in the form of flattened sacs (or cistern) or of

tubules. Because of the presence of the endoplasmic reticulum the cytoplasm is

divided into two components, one within the channels and one outside them.

In most places the membranes forming the endoplasmic reticulum are studded with

minute particles of RNA called ribosomes. The presence of these ribosomes gives

the membrane a rough appearance. Membranes of this type form what is called the

rough (or granular) endoplasmic reticulum. In contrast some membranes are

23

devoid of ribosomes and constitute the smooth or agranular endoplasmic reticulum. Rough endoplasmic reticulum represents the site at which proteins are

synthesized. The attached ribosomes play an important role in this process. Smooth

endoplasmic reliculum is associated with numerous biochemical processes in-

cluding carbohydrate metabolism. Products synthesized by the endoplasmic

reticulum are stored in the channels within the reticulum. Ribosomes, and

enzymes, are present on the 'outer' surfaces of the membranes of the reticulum.

Golgi Complex The Golgi complex (or Golgi apparatus) can be seen as a small structure of

irregular shape, usually present near the nucleus. When examined with the EM the

complex is seen to be made up of membranes similar to those of smooth

endoplasmic reticulum. The membranes form the walls of a number of flattened

sacs that are stacked over one another. Towards their margins the sacs are

continuous with small rounded vesicles. The Golgi complex is intimately

connected with the formation of several secretory products, specially those

containing carbohydrates.

The protein component of these products is synthesized in rough

endoplasmic reticulum. As the proteins pass through successive sacs of the Golgi

complex they undergo a process of purification. In the Golgi complex

carbohydrates are added to the proteins to form protein-carbohydrate complexes.

These complexes are formed within the cisternae of the Golgi apparatus. They pass

to the margins of the cisternae where they separate from the Golgi complex

forming membrane bound secretory vacuoles.The membranes of the Golgi

complex give attachment to enzymes associated with carbohydrate synthesis.

Lysosomes may also be produced in the Golgi complex. Lysosomes

These vesicles (primary, secondary, rest bodies and autophagosomes)

contain enzymes that can destroy unwanted material present within a cell. Such

material may have been taken into the cell from outside (e.g., bacteria); or may

represent organelles that are no longer of use to the cell. The enzymes present in

lysosomcs include proteases, Lipases, curbohydrases, and acid phosphatase. (As

24

many as 40 different lysosomal enzymes have been identify. Passing along the

channel of the reticulum they reach the Golgi complex. Here the enzymes come to

be surrounded by membranes and are set free into the cyloplasm in the form of

vesicles that bud off from marginal areas of the Golgi complex.

Lysosomes help in 'digesting' the material within phagosomes (described

above) as follows. A lysosome, containing appropriate enzymes, fasts with the

phagosome so that the enzymes of the former can act on the material within the

phagosome. These bodies consisting of fused phagosomes and lysosomes arc

refried to a secondary lysosomes or phagolysosomes. In a similar manner lysosomes may also fuse with pinocytotic vesicles. The

structures formed by such fusion often appear to have numerous small vesicles

within them and called multivesicular bodies. After the material in phagosomes or pinocytotic vesicles has been 'digested'

by lysosomes, some waste material may be left. Some of it is thrown out of the cell

by exocytosis. However, some material may remain within the cell in the form of

membrane bound residual bodies. Lysosomal enzymes play an important role in

the destruction of bacteria phagocytosed by the cell. Lysosomal enzymes may also

be discharged out of the cell and may influence adjoining structures. Lysosomes

are present in all cells except mature erythrocytes. They are a prominent feature in

neutrophil leucocytes.

Ribosomes We have seen above that ribosomes are present in relation to rough

endoplasmic reticulum. They may also lie free in the cytoplasm. They may be

present singly in which case they are called monosomes; or in groups which are

referred to as potyribosomes (or polysomes). Each ribosome consists of proteins

25

and RNA (ribonucleic acid) and is about 15 nm in diameter. The ribosome is made

up of two subunits one of which is larger than the other. Ribosomes play an

essential role in protein synthesis.

Microfilaments & Microtubules

The cytoplasm of many varieties of cells contains thin elongated elements.

Some of these are tubular, and are circular in cross section: they are called

microtubules. Others, called micro filaments, are solid fibres. These elements can

be made out in light microscopic preparations of dividing cells in which they form

the mitotic spindle. With the EM they can be identified in many other cells and the

distinction between tubule and filaments can also be made out. Microtubules and

microfilaments (along with some other filaments present in the cytoplasm)

constitute the cytoskeleton.

Both microtubules and microfilaments are made up of proteins. The proteins

forming microtubules are called tubulins. Microfilaments are usually composed of

a protein called actin, but in some situations (e.g., in neurons) they may be

composed of other proteins. A microtubule is about 24 nm in diameter. A microfilament is 6-8 nm in

diameter. Intermediate filaments about 10 nm in diameter are found in many cells

where structural strength is required: these include nerve cells (in which they are

seen as neurofilaments), epithelial cells and neuroglial cells.

26

Centrioles

All cells capable of division (and even some which do not divide) contain a

pair of structures called centrioles. With the light microscope the two centrioles are

seen as dots embedded in a region of dense cytoplasm which is called the

centrosome. With the EM the centrioles are seen to be short cylinders that lie at

right angles to each other. When we examine a transverse section across a

centrioles (by EM) it is seen to consist essentially of a series of microtubules

arranged in a circle. There are nine groups of tubules each group consisting of

three tubules. Centrioles play an important role in the formation of various cellular

structures that are made up of microtubules. These include the mitotic spindles of

dividing cells, cilia, flagella, and some projections of specialized cells (e.g., the

axial filaments of spermatozoa). It is of interest to note that cilia, flagella and the

tails of spermatozoa all have the 9+2 configuration of microtubules that are seen in

centrioles.

SPETIAL ORGANELLES Many cells show projections from the cell surface. The various types of

projections are described below. Cilia These can be seen, with the light

microscope, as minute hair-like projections from the free surfaces of some

epithelial cells. In the living animal cilia can be seen to be motile. Details of their

structure, described below, can be made out only by EM. The free part of each

cilium is called the shaft. The region of attachment of the shaft to the cell surface is

called the base (also called the basal body, basal granule, or kinetosome). The free

end of the shaft tapers to a tip. In structure the cilium consists of an outer covering

which is formed by an extension of the cell membrane; and an inner core that is

formed by microtubules arranged in a definite manner. It has a striking similarity

to the structure of a centriole (described above). There is a central pair of tubules

which is surrounded by nine pairs of tubules. The outer tubules are connected to

the inner pair by radial structures (which are like the spokes of a wheel). Other

projections pass outwards from the outer tubules. As the tubules of the shaft are

traced towards the tip of the cilium it is seen that one tubule of each outer pair ends

short of the tip so that near the tip each outer pair is represented by one tubule

only. Just near the tip, only the central pair of tubules is seen. At the base of the

27

cilium one additional tubule is added to each outer pair so that here the nine outer

groups of tubules have three tubules each, exactly as in the centriole. Functional

significance of cilia The cilia lining an epithelial surface move in coordination

with one another the total effect being that like a wave. As a result fluid, mucous,

or small solid objects lying on the epithelium can be caused to move in a specific

direction. Movements of cilia lining the respiratory epithelium help to move

secretions in the trachea and bronchi towards the pharynx. Ciliary action helps in

the movement of ova through the uterine tube, and of spermatozoa through the

male genital tract. In some situations there are cilia-like structures that perform a

sensory function. They may be non-motile, but can be bent by external influences.

Such 'cilia' present on the cells in the olfactory mucosa of the nose are called

olfactory cilia: they are receptors for smell. Similar structures called kinocilia are

present in some parts of the internal ear. In some regions there are hair-like

projections called stereocilia: these are not cilia at ah1, but are large microvilli.

Flagella These are somewhat larger processes having the same basic structure as

cilia. In the human body the best example of a flagellum is the tail of the sper-

matozoon. The movements of flagella are different from those of cilia. In a

flagellum movement starts at its base. The segment nearest the base bends in one

direction. This is followed by bending of succeeding segments in opposite

directions so that a wave like motion passes down the flagellum. When a

spermatozoon is suspended in a fluid medium this wave of movement propels the

spermatozoon forwards (exactly in the way a snake moves forwards by a wavy

movement of its body). Microvilli These are finger-like projections from the cell

surface that can be seen only with the EM. Each microvillus consists of an outer

covering of plasma membrane and a cytoplasmic core in which there are numerous

microfilaments. Numerous enzymes have been located in microvilli. With the light

microscope the free borders of epithelial cells lining the small intestine appear to

be thickened: the thickening has striations perpendicular to the surface. This

striated border of light microscopy has bcc n shown by EM to be made up of long

microvilli arranged parallel to one another. In some cells the microvilli are not arranged so regularly. With the light

microscope the microvilli of such cells give the appearance of a brush border.

Microvilli greatly increase the surface area of the cell and are, therefore,

seen most typically at sites of active absorption e.g., the intestine, and the proximal

and distal convoluted tubules of the kidneys. Modified microvilli called stereocilia

are seen on receptor cells in the internal ear, and on the epithelium of the

epididymis. Cell inclusions Non-living, non-participating, poorly structured cell

elements, very rarely seen in an intra-nuclear position; usually cytoplasmic.

3.3. Literature recommended

Main Sources:

1. L.C. Junqueira, J. Carneiro - “Basic histology” – 11 edition - 2005. 2. A.S. Pacurar, J.W. Bigbee – “Digital histology” – Verginia - 2004. 3. “Color Atlas of basic histology” – R.Berns – 2006.

28

4. Sadler T.V. – “Medical embryology” Montana – 1999. 5. Ronald W., Dudek Ph.D. –“Embryology” 2 edition – 1998. 6. Inderbir Singh Textbook of Human Histology.- Jaypee. India. - 1997. 7. Ten Cate A.R. Oral Histology.- St.Louis, Baltimore, Toronto.- 1995.

8. William A., Beresford M.A. Cytology and Histology.- Anatomy department, West Virginia University, USA.- 1992.

9. K.E. Jonson - “Histology and cell biology” – 2 edition – Washington – 1991.

Additional ones:


3.4 How to work with the literature recommended:












To be ready to give an answer to the

following:


A. Questions to be answered:

1. What is the cytoplasm?

2. Give the classification of the organelles?

3. Describe a structure of the mitochondria?

4. Give the characteristics of the endoplasmic reticulum?

5. What is the Golgi complex? 6. Describe the structure of lysosomes?

7. Give the characteristics of the ribosomes? 8. Describe the structure of microfilaments and microtubules? 9. Give the characteristics of centrioles? 10. Describe the structure of the cilia? 11. Give the characteristics of flagella? 12. Describe the structure of the microvilli? 13. Give the characteristics of cell inclusions?

B. Test tasks to be done: See enclosure.



2) Work with the tables and microscope.


4) To sketch in an album the investigated preparations.






29


- “Principal causes of lysosomal diseases development”. Subject Histology, cytology and embryology



Topic 4 NUCLEUS. DIVISION OF CELLS. CELL’S CYCLE Course 1

Faculty Dental

Hours: 2

1. The topic basis: the topic “Cell nucleus. Division of cells” is very important for





1) To have general knowledge of the topic studied;

2) To understand, to remember and to use the knowledge received;

3) To learn the classification, structure, functions of the Nucleus;


authorizing it;






Med. Biology the structure of the cells and

tissues

work with a light

microscope



work with a light

microscope

Organic Chemistry the chemical content of the

cell

Speak of the topic

3.2. The contents of the topic:

The nucleus constitutes the central, more dense part of the cell. It is usually

rounded or ellipsoid. Occasionally it may be elongated, indented or lobed. It is

usually 4-10 um in diameter. The nucleus contains inherited information which is

necessary for directing the activities of the cell as we shall see below.

Nuclear constituents

1. Chromatin - mainly DNA and dispersed at interphase (except for one female X chromosome); has a fine granular appearance in routine EM, but is

fibrillar; some RNA is present.

2. Nucleolus/nucleoli - dense clumped granules; nucleic acid is mainly RNA, but some DNA is there. Perinucleolar/nucleolus-associated chromatin has a

special relation to the nucleolus. A range in the number of nucleoli present

usually exists, e.g., in the liver cells with one nucleus, 1 to 6, with 2 the

30

modal value. Removal of DNA by DNase allows one to see nucleoli in the

very dense nuclei of lymphocytes and some other cells.

3. Nuclearmembrane – a double layered membrane that surrounded a nucleus. It content the nuclear pores.

4. Nucleoplasma – the fluid spaces between the various constituents of the nucleus.

Histological methods for the nucleus:

o Nuclear structure is seen in TEM.

o Radioautography for rates of cell division uses tritium-labelled

precursors of nucleotides, e.g., thymidine and cytidine.

Bromodeoxyuridine (BrdU) is incorporated in place of thymidine, so

that cells synthesizing DNA prior to division can be revealed by a

labelled antibody to the BrdU.

o Chromosomes and their banding patterns are studied in cytogenetics.

o Fluorescent in-situ hybridisation uses 'coloured' nucleotide probes to

identify a particular chromosome and, on it, the gene of clinical

interest - normal, mutated, translocated, deleted, duplicated, truncated,

etc.

o Silver staining reveals the nucleolar organizer regions (NORs) as

intranuclear black dots, because it marks the acidic proteins binding to

the genes coding for ribosomal RNA located on certain chromosomes.

An increase in the number of NORs so shown (AgNORs) correlates

31

with dysplastic (abnormal) growth, and may indicate malignant

tendencies in epithelial cells.

Chromatin

In recent years there has been a considerable advance in our knowledge of the

structure and significance of chromatin. It is made up of a substance called

deoxyribonucleic acid (usually abbreviated to DNA); and of proteins. Most of the

proteins in chromatin are histones. During cell division the entire chromatin within

the nucleus becomes very tightly coiled and takes on the appearance of a number

of short, thick, rodlike structures called chromosomes. Chromosomes are made up

of DNA and proteins. Proteins stabilize the structure of chromosomes.

In usual classroom slides stained with haematoxylin and eosin, the nucleus stains

dark purple or blue while the cytoplasm is usually stained pink. In some cells the

nuclei are relatively large and light staining. Such nuclei appear to be made up of a

delicate network of fibres: the material making up the fibres of the network is

called chromatin (because of its affinity for dyes). At some places (in the nucleus)

the chromatin is seen in the form of irregular dark masses that are called

heterochromalin (condensering). At other places the network is loose and stains

lightly: the chromatin of such areas is referred to as euchromatin

(decondensering). Nuclei which are large and hi which relatively large areas of

euchrouiatin can be seen are referred to as open-faced nuclei. Nuclei which are

made up mainly of heterochiomatin are referred to as closed-face nuclei.

In addition to the masses of heterochromatin (which are irregular in outline), the

nucleus shows one or more rounded, dark staining bodies called nucleoli. The

nucleus also contains various small granules, fibres and vesicles (of obscure

function). The spaces between the various constituents of the nucleus described

above are filled by a base called the nucleoplasm.

Nuclear membrane With the EM the nucleus is seen to be surrounded by a double layered nuclear

membrane. The outer layer of the nuclear membrane is continuous with

32

endoplasmic reticulum. The inner layer provides attachment to the ends of

chromosomes. Deep to the inner membrane there is a layer containing proteins and

a network of filaments: this layer is called the nuclear lamina. At several points the

inner and outer layers of the nuclear membrane fase leaving gaps called nuclear

pores. Each pore is surrounded by dense protein: the region of dense protein and

the pore together - form the pore complex.

Nuclear pores represent sites at which substances can pass from the nucleus to the

cytoplasm and vice versa. The nuclear pore is about 80 nm across. It is partly

covered by a diaphragm which allows passage only to particles less than 9 nm in

diameter. A typical nucleus has 3000 to 4000 pores.

33

Nucleoli We have seen that nuclei contain one or more nucleoli. They stain intensely with

basic dyes like haematoxylin. In ordinary preparations they can be distinguished

from heterochromatin by their rounded shape. (In contrast masses of

heterochromatin are very irregular).

Using histochemical procedures that distinguish between DNA and RNA it is seen

that the nucleoli have a high RNA content. With the EM nucleoli are seen to have a

central filamentous zone (pars filamentosa) and an outer granular zone (pars

granulosa) both of which are embedded in an amorphous material (pars

amorphosa). Nucleoli are formed in relationship to the secondary constrictions of specific

chromosomes. These regions are considered to be nucleolar organizing centres.

Parts of the chromosomes located within nucleoli constitute the pars chromosoma

of nucleoli.

Nucleoli are sites where ribosomal RNA is synthesized. The templates for this

synthesis are located on the related chromosomes. Ribosomal RNA is at first in the

form of long fibres that constitute the fibrous zone of nucleoli. It is then broken up

into smaller pieces that constitute the granular zone. Finally, this RNA leaves the

nucleolus, passes through a nuclear pore, and enters the cytoplasm where it takes

part in protein synthesis.

Multiplication of cells takes place by division of pre-existing cells. Such

multiplication constitutes an essential feature of embryonic development. Cell

multiplication is equally necessary after birth of the individual for growth and for

replacement of dead cells. We have seen that the chromosomes within of nuclei of cells carry genetic

information that call trolls the development and functioning of vat rows cells and

tissues — and, therefore, of the body as a whole. When a cell divides it is essential

that the whole of the genetic information within it be passed on to both the

daughter cells resulting from the division. In other words the daughter cells must

34

have chromosomes identical in number and in genetic content to those in the

mother cell. This type of cell division is called mitosis. A different kind of cell division called meiosis occurs during the formation of

gametes. This consists of two successive divisions called the first and second

meiotic divisions. The cells resulting from these divisions (i.e., the gametes) differ

from other cells in the body in that the number of chromosomes is reduced to half

the normal number, and the genetic information in the various gametes produced is

not identical. Mitosis

Many cells of the body have a limited span of functional activity at the end

of which they undergo division into two daughter cells. The daughters cells hi turn

have their own span of activity followed by another division. The period during

which the cell is actively dividing is the phase of mitosis. The period between two

successive divisions is called the interphase.

The greater part of interphase is called the G1 stage, which may last from a

few hours to many years. During this period the cell carries out its 'normal'

functions. Protein synthesis takes place mainly in this phase. About 12 hours

before the onset of mitosis the synthesis of DNA takes place and is completed in

about 7 hours: this period is called the S stage (S for synthesis). The last five hours

before mitosis are utilized for synthesis of proteins required for cell division. This

is called the G2 stage of interphase.

Mitosis is conventionally divided into a number telophase. At this stage each

35

chromosome consists of stages called prophase, metaphase, anaphase and

telophase. The sequence of events of the mitotic cycle is best understood by

starting with a cell in thelophase. With the progress of telophase the chromatin of

the chromosome uncoils and elongates and the chromosome can no longer be

identified as such.

However, it is believed to retain its identity during the interphase (which

follows telophase). During the S stage of interphase the DNA content of the

chromosome is duplicated so that another chromatid identical to the original one is

formed: the chromosome is now made up of two chromatids. When mitosis begins

(i.e., during the prophast) the chromatin of the chromosome becomes gradually

more and more coiled so that the chromosome become recognizable as a thread-

like structure that gradually acquires a rod-like appearance.

While the changes described above are occurring in the chromosomes a

number of other events are taking place. The two centrioles separate and move to

opposite poles of the cell. They produce a number of microtubules that pass from

one centriole to the other and form a spindle. Tubules radiating from each centriole

create a star like appearance or aster.

The spindle and the two asters collectively form the diasler (also called

36

amphiaster or achromatic spindle). Meanwhile the nuclear membrane breaks down

and the nucleoli disappear. With the formation of the spindle the chiomosomes

move to a position midway between the two centrums u c., at the equator of the

cell) where each chromosome becomes attached to microtubules of the spindle by

its centromere. This stage is referred to as metaphase. The plane along which the

chromosomes lie during metaphase is the equatorial plate.

In the anaphase the centromere of each chromosome splits longitudinally

into two so that the chromatids now become independent chromosomes. At this

stage the cell can be said to contain 46 pairs of chromosomes. One chromosome of

each such pair now moves along the spindle to either pole of the cell. This is

followed by telophase in which two daughter nuclei are formed by appearance of

nuclear membranes around them. The chromosomes gradually elongate and

become indistinct. Nucleoli reappear. The centriole is duplicated at this stage or in

early interphase.

The division of the nucleus is accompanied by the division of the cytoplasm.

In this process the organelles are presumably duplicated and each daughter cell

comes to have a full complement of them. The rate of cell division varies from tissue to tissue being greatest fit those

epithelia which lose cells because of friction (e.g., the epidermis and the lining

cells of the intestine). The rate varies with demand becoming much greater during

repair after injury. The rate is precisely controlled to correlate with demand.

Failure of such control results in uncontrolled growth leading to formation of

tumours. Various abnormalities in mitosis may be produced by exposure to various

radiations, the most important being nuclear radiation. Mitosis can be arrested by

chemicals. One of them — colchicin (or colcemide) — stops mitosis at metaphase

and allows us to study chromosomes at this stage. Meiosis

As already stated meiosis consists of two successive divisions called the first

and second meiotic divisions. During the interphase preceding the first division

duplication of the DNA content of the chromosomes takes place as in mitosis.

37

First Meiotic Division The prophase of the first meiotic division is prolonged and is usually divided

into a number of stages as follows. (a) Leptotene: The chromosomes become

visible (as in mitosis). Although each chromosome consists of two chromatids

these cannot be distinguished at this stage. At first the chromosomes are seen as

threads bearing bead-like thickenings (cliromomeres) along their length. One end

of the thread is attached to the nuclear membrane. During leptotene the

chromosomes gradually become thicker and shorter. (b) Zygotenc: We have seen

that the 46 chromosomes in each cell consist of 23 pairs (the X and Y

chromosomes of the male being taken as a pair). The two chromosomes of each

pair come to lie parallel to each other, and are closfcly ap-posed. This pairing of

chromosomes is also referred to as synopsis or conjugation The two

chromosomes together constitute a bivalent. (c) Pachytene: The two chromatids

of each chromosome become distinct. The bivalent now has four chromatids in it

and is called a tetrad. There are two central and two peripheral chromatids,

one from each chromosome. An important event now takes place. The two central

chromatids (one belonging to each chromosome of the bivalent) become coiled

over each other so that they cross at a number of points. This is called crossing

over. At the site where the chromatids cross they become adherent: the points of

adhesion are called chiasmata. (d) Diplotene: The two chromosomes of a bivalent

now try to move apart. As they do so the chromatids 'break' at the points of

crossing and the 'loose' pieces become attached to the opposite chromatid. This

results in exchange of genetic material between these chromatids. A study of that

as a result of this crossing over of genetic material each of the four chromatids of

the tetrad now has a distinctive genetic content.The metaphase follows. As in

mitosis the 46 chromosomes become attached to the spindle at the equator, the two

chromosomes of a pair being close to each other.The anaphase differs from that in

mitosis in that there is no splitting of the centromeres. One entire chromosome of

each pair moves to each pole of the spindle. The resulting daughter cells, therefore,

have 23 chromosomes, each made up of two chromatids.The telophase is similar to

that in mitosis.The first meiotic division is followed by a short interphase. This

differs from the usual interphase in that there is no duplication of DNA. Such

duplication is unnecessary as the chromosomes of the cells resulting from the first

meiotic division already possess two chromatids each.

Second Meiotic Division The second meiotic division is similar to mitosis. However, because of the

crossing over that has occurred during the first division, the daughter cells are not

identical in genetic content. This is the reason for regarding it as a meiotic division. At this stage it may be repeated that the 46 chromosomes of a cell consist of

23 pairs, one chromosome of each pair being derived from the mother and one

from the father. During the first meiotic division the chromosomes derived from

the father and those derived from the mother are distributed between the daughter

cells entirely at random. This, along with the phenomenon of crossing over, results

in thorough shuffling of the genetic material so that the cells produced as a result

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of various meiolic divisions (i.e., ova and spermatozoa) all have a distinct genetic

content. A third step in this process of genetic shuffling takes place at fertilization

histology.umsa.edu.ua · 2020. 12. 31. · 1 subject histology, cytology and embryology modul №1...

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